Fluoride is added to drinking water supplied to two-thirds of the communities in the U.S. because of its proven benefit for reduction of dental caries. However, exposure to elevated dietary fluoride during tooth development leads to the enamel structural defect called enamel fluorosis, with dental aesthetic concerns in greater than 12% of residents of some fluoridated communities. Elevated fluoride in drinking water provided to rats led to alterations in the normal ameloblast morphology, and effects on these fluoride-sensitive, enamel-secreting cells are thought to contribute to the enamel structural defects. Using tooth organ culture and specific inhibitors, we have shown that the RhoA pathway is responsible for one of the responses of ameloblasts to sodium fluoride, that of elevation of filamentous actin (F-actin). F-actin localization in ameloblasts normally changes during developmental stages as ameloblast function changes, and we propose that RhoA is central to fluoride-induced cytoskeletal deregulation. We propose to test the central position of the RhoA pathway in the fluoride response with 2 specific aims. In Aim 1, a novel transgenic mouse model will be generated that expresses a dominant-negative RhoA in ameloblasts, the epithelial cell layer that secretes enamel. Histological, confocal and scanning electron microscopic images of wild-type and transgenic mice will be evaluated, and the cellular response to fluoride in drinking water will be analyzed in vivo. In Aim 2, tooth organs from wild-type and RhoADN transgenic mice will be cultured with Rho pathway inhibitors or an activator and analyzed by confocal microscopy and biochemical approaches to monitor the response of RhoA and downstream targets in the pathway to fluoride. These experiments will provide a new animal model for discovery of the mechanism of action for this environmental agent on a small G protein signaling pathway. Fluoride is commonly added to public drinking water, but there are geographic locations where the maximum recommended dose is naturally exceeded. Although elevated fluoride levels lead to an unsightly dental defect known as fluorosis, slightly lower levels are highly beneficial in reducing dental caries. A better understanding of the health-related benefits and problems associated with environmental fluoride will lead to more informed policy decisions and fundamental knowledge about the impact of this environmental agent. [unreadable] [unreadable] [unreadable]